Faster, Cooler DNA gels
All over the world, molecular biologists are tragically wasting hours of their life running DNA gels using tris-based conduction buffers like TBE or TAE.
These buffers are known to overheat at high voltages, causing problems with gel integrity, sample denaturation and more. Because of this, molecular biologists are forced to keep the voltage of their gels to a maximum of 5-10 volts/cm (e.g 100 volts for a 10 cm gel) and extend the running time, sometimes to hours.
Although long gel runs, like long restriction digests, are often used as a convenient coffee break opportunity they can also eat into the molecular biologist’s precious time, leading to longer and less efficient working days.
But, in 2004, a team of scientists from Johns Hopkins came up with solutions (pardon the pun) to this problem. They have developed and verified three conductive buffers that stay cool during electrophoresis, allowing the voltage to be racked up to a massive 35 volts/cm without any problem, reducing the time taken to run gels by up to 7 times.
Between them, the three buffers cover all of the molecular biologist’s DNA gel needs. The buffers are:
- 10mM sodium boric acid (Na2B4O7/Borax)
For standard applications (separation of DNA fragments from 100bp-5kbp). - 5mM lithium acetate (LiOOCCH3, CAS:546-89-4)
For separation of fragments longer than 3 kbp. - 1mM lithium boric acid (Li2B4O7, CAS:12007-60-2)
For separating small DNA fragments and ssDNA
Our lab’s standard DNA gel buffer has become 40 g of Borax into 1 l of water which gives a 20x stock solution. Small gels run in 10-15 minutes at 200V.
The sodium borate and lithium acetate buffers can also be used for RNA gels in place of MOPS buffer.
Simply make up the required buffer, use the same buffer in the gel and the tank, turn up the voltage to 10-35 volts/cm and watch that DNA go.
Despite this excellent work, there are still thousands of molecular biologists who have not yet been shown that there is an alternative to wasting their precious hours using tris buffered gels.
Help us to eliminate this tragedy by spreading the word about this method for faster, cooler DNA gels. You can do this by clicking the link below this article to e-mail it to your molecular biologist friends. We, and they, thank you for your help.
Further reading:
1. Brody and Kern(2004) Biotechniques 36 p214
2. Brody et al (2004) Biotechniques 37 p598 (Free registration required)
3. Hudson, Biocompare protocols (a protocol giving a quick overview)
Photo: Klis
molecular biology
I want to add that the new buffers (which really are old, but just haven’t been used for agarose gels before) also give you cleaner bands. In addition, SBA is cheaper than TBE/TAE.
You can also prepare this buffer by making up 10mM NaOH and adjusting the pH to 8.5 with boric acid powder. We routinely run our gels at 150v, and sometimes at 300v although the gel chambers don’t like it very much. The drop well systems seem to handle the higher voltages much better. Perhaps even run it in the cold room. I know in one part of the papers above, they resolved nucleic acid at 1000v in 5 minutes.
I tried running my gel with lithium acetate buffer at 300v but i observed the heating effect and it did not turn well!I’ve attached the picture below.Any suggestions
20x Lithium Acetate: 6.6g lithium acetate, Made up to 1 L with water.
http://img329.imageshack.us/img329/1927/highvoltagerunvr5.png
Tried this ~ 2 years ago and, whilst succesful at running gels without overheating in < 15 min, the bands were definitely more smeared and less sharp than a typical ’slow’ TAE/TBE gel. In the original paper the bands look fine but I was unable to reproduce that at the higher voltages. In my experience: useful to check quickly if a PCR has worked but not something I’d put in a thesis/paper.
CK: Yes, a gel at 300V is worse than a 80V-TBE-gel, I agree. For nice gels, I’d slow them down, to 150V, for instance. SBA is still the better, cheaper generic buffer in my opinion, if run at the same voltage as TBE.
Ok, perhaps I should have suggested this first. We normally depending on the amount of DNA loading dye added to a well, start the gel at 100V, then proceed to higher voltages, this normally works well.
Of course if you’re filling your gel wells to the top, things don’t run very well, but if you have bigger wells, and are able to add more loading dye to make it sink better, or can load a smaller volume, it generally gives better resolution. If nothing else, run this buffer at your normal voltages, it is cheaper than the normal buffers.
Does anyone know how well these gels work with the typical gel extraction protocols?
i’ve never had any problems with extracting a band from my gels and I don’t see why the buffer would make any difference for the gel extraction.
I’ve thought about trying these buffers. Isn’t it the basis of the flash gel and invitrogen precast (EZgel?) systems? I read that it doesn’t work well with restriction digests, something about the salt concentration of the digests not being compatible. Has anyone tried this?
Here’s a link to a FAQ from a company selling the buffers…
http://www.fasterbettermedia.com/technologyfaq/faq.html
Thanks for the link Caroline! The FAQ says that if you use HIGH salt restriction digest buffers, you’d better dilute them a little before putting them onto a gel. As SBA/LB are lower salt than TBE/TAE, the salt difference samples/gel starts to play a role.
It seems that I never used high salt buffers until now with the gels so I cannot say how strong the effect is.
I guess it is a good idea in general, also for TBE, to dilute the ladder with the buffer of the samples you’re running and, in the case of LB/SBA, to dilute the samples with the running buffer a bit.
Has anyone experienced this? Has anyone calculated which of the standard buffers (e.g. NEB 1-4) has a salt content > 50 mM ?